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The discovery more than a century ago that plants possess major genes for resistance to pathogens (Biffen, 1905) has been of immense importance in biology and agriculture. A few years before Biffen's discovery, researchers in France had demonstrated that plants could be immunized against virulent pathogens by previous inoculation with nonpathogenic symbionts (Beauverie, 1901; Ray, 1901). The latter phenomenon has been variously known as acquired physiological immunity (Chester, 1933), cross-protection and induced resistance (Sequeira, 1984), and the inducers include symbionts such as mycorrhizal fungi and root endophytes (Van Wees et al., 2008). It is now clear that resistance induced by non-pathogenic symbionts can result in expression of genetic resistance in host plants (Wang et al., 2005). Thus, the separate discoveries at the turn of the twentieth century have, in a sense, now been integrated.
However, that integration is far from complete. The genetic resistance that is induced by symbionts is likely to be quantitative because major genes are typically induced only by specific pathogens (Jones & Dangl, 2006). Yet, both ecologists (Price et al., 2004) and plant breeders (Clair, 2010) still regard quantitative resistance as the product of minor genetic interactions between host and pathogen, influenced primarily by the abiotic environment. Foliar endophytes have largely been ignored, both theoretically and practically.
This is an important shortcoming because endophytic symbionts are known to enhance disease resistance in some manner (Arnold et al., 2003; Ganley et al., 2008), and foliar endophytes are also a common and diverse part of the biotic environment of plants. Endophytes might induce resistance locally or systemically (Bailey et al., 2006), but they might also directly interact with a pathogen. The extent of induction and of direct interaction could each vary with endophytes, and this inference raises many questions given the diversity of endophytes (Ganley et al., 2004; Arnold, 2007), particularly in leaves.
Of the three, nonclavicipitaceous classes of endophytes in plants, it is the shoot-restricted, largely foliar endophytes of ‘class 3’ that are the most diverse and least understood ecologically (Rodriguez et al., 2009). Although some foliar endophytes are known to enhance resistance in some plants, their specific contribution to quantitative resistance has not been determined.
In Populus affected by Melampsora rust, quantitative (Lefevre et al., 1998; Dowkiw et al., 2003) and major-gene resistance (Newcombe et al., 1996, 2001) can be distinguished by phenotype. Observation readily indicates that endophytes do not induce hypersensitive phenotypes typical of major genes in this system. As major genes may provide complete resistance it is necessary to pair host genotypes with virulent pathotypes of Melampsora in order to observe quantitative resistance (Flor, 1971; Van der Plank, 1984). This is the first condition for the determination of the contribution of endophytes to quantitative resistance.
The second condition is to ensure that both independent variables (i.e. endophytes on the one hand and combined host/pathogen genotypes on the other) actually vary. This can be achieved easily for endophytes by choosing distinct taxa from among the diverse foliar endophytes of Populus. However, combined host/pathogen genotypes are more challenging; distinct poplar genotypes need to be specifically distinguished in terms of the combined resistance and virulence genes of both host and pathogen, respectively. Such combinations have been termed interorganismal genotypes, in the sense of Loegering (1978). As defeated major genes can contribute to quantitative resistance (Dowkiw & Bastien, 2007), these genes should be included in the determination of these combined genotypes.
In the case of virulent Melampsora on Populus, quantitative resistance is known to vary continuously and it is commonly quantified using estimates of uredinial density (UD) and latent period (LP) (Newcombe, 1998; Newcombe et al., 2001). However, UD is more commonly employed as a more direct measure of quantitative resistance than LP. Endophytes are commonly isolated from both rusted and non-rusted poplar leaves.
Here, we varied endophytes associated with poplar leaf rust simply by isolating four distinct taxa. We then showed that six poplar genotypes were distinguishable when inoculated with Melampsora in terms of the combined expression of resistance and virulence. A total of 30 experimental combinations were thus available: four endophytes (plus their control) and six host/pathogen genotypes. We were then able to determine the extent to which these two variables (i.e. endophyte and host/pathogen genotype) explain quantitative variation in resistance to virulent Melampsora rust, the most important foliar pathogen of Populus (Newcombe, 1996). Finally, although it can be difficult to distinguish between the mechanism of local induction and that of direct interaction (Van Loon et al., 1998), and we did not attempt that here, we did determine whether endophyte effects were local or systemic. Local effects were contrasted with systemic effects that were expected to develop in younger leaves after the endophyte inoculations.
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Foliar endophytes were surprisingly important contributors to quantitative resistance to leaf rust in our experimental system. Four foliar endophytes explained 54% of the variation in quantitative resistance (UD) among six poplar genotypes varying in genetic resistance to virulent isolates of Melampsora × columbiana. The local, within-leaf effects were strong enough that further investigation is needed to determine if foliar endophytes are the second line of defense behind major genes for resistance to leaf rust.
Even though the foliar endophytes of this study did not induce resistance systemically, they may have induced it locally (Van Wees et al., 2008). A competing explanation for the local effects of endophytes is direct interaction with Melampsora. Either mechanism could be compatible with the observed constant ranking of the four endophytes (i.e. Stachybotrys, Trichoderma, Ulocladium and Truncatella, in that order in terms of magnitude of effect; Tables 5, S2, S3), regardless of combined host/pathogen genotypes. It is extremely improbable [1/(4!)5 c. 1 in 8 00 000] that such constant ranking is due to chance. For local induction of host resistance to be compatible with constant ranking, the four endophytes would have to vary in their capacity to induce yet be insensitive to the differences in combined host/pathogen genotypes employed in this study. By contrast, direct interaction between endophytes and Melampsora could explain constant endophyte ranking without having to also account for interactions with combined host/pathogen genotypes.
It was essential for these inferences that the poplar genotypes were specifically distinguishable in terms of genes for resistance. If we had simply selected different genotypes of poplar without testing their resistance genes, we would not have been sure that there was actually variation in resistance genes. Just as otherwise distinct people could have the same genes for blood type, otherwise distinct, poplar genotypes could have the same resistance genes. In terms of major genes, genotypes 1 and 5 were identical in our tests in that they lacked resistance to the four pathotypes. Genotypes 3 and 6 were also identical as RRSRRS patterns were found for both. However, in terms of quantitative, genetic resistance (Fig. 2) host/pathogen genotype 1 was distinguished from genotype 5 and host/pathogen genotype 3 from genotype 6. In other words, when both major-gene and quantitative resistance were considered, each of the six host/pathogen combinations was a distinct genotype. Yet none of this host/pathogen variation affected the ranking of the four endophytes.
Endophyte infection may enhance disease suppression whether the mechanism involves local induction of host resistance or direct interaction with the pathogen (Mejia et al., 2008; Lee et al., 2009). Stachybotrys and Trichoderma had the strongest effects on uredinial density and they infected to the greatest extent, as measured by reisolation. Ulocladium and Truncatella had weaker effects on uredinial density and more poorly developed abilities to infect, although each was able to infect their source host, P. trichocarpa (genotype 6), with the expected stronger effects associated with colonization. In general, the effects of endophytes were strongest in P. trichocarpa, although effects were almost as strong in the five hybrids (Figs S1, S2). Therefore, if anything, this study may have underestimated the magnitude of endophyte effects by coupling endophytes isolated from P. trichocarpa with five hybrid genotypes.
In this study, four, specific endophytes contributed to quantitative resistance in individual leaves of six combined poplar/rust genotypes. However, overall foliar endophytes must be highly variable in nature at the level of a tree. Each poplar tree is likely colonized by diverse endophytes that vary from leaf to leaf (Santamaria & Diez, 2005; Albrectsen et al., 2010) and that may also vary among host genotypes. Overall, foliar endophytes of Populus are likely to vary more widely in their abilities to reduce rust severity than the four of this study. In other words, endophytes with stronger effects than Stachybotrys will likely be isolated from Populus; similarly, endophytes with no significant effect, or even a rust-enhancing effect will also likely be found. Furthermore, young foliage is likely to be uncolonized by endophytes (Stone, 1987; Arnold et al., 2003). Collectively, these likelihoods make it probable that the foliage of a tree is a highly variable mosaic with respect to endophyte-mediated resistance. This is quite unlike the major-gene, minor-gene and systemically induced forms of defense that should not vary within the foliage of a tree.